Aedes aegypti transmits a number of arboviral diseases including dengue fever, which threatens virtually one half of the human population. Recent physical and genetic mapping efforts assigned 71% of the original draft genome assembly to the mosquito chromosomes. However, the Ae. aegypti genome assembly still remains highly fragmented. Moreover, both mapping approaches determined high rates of misassembly in the existing genomic supercontigs. The lack of a high-quality genome assembly has hindered population and evolutionary genomics studies. Two subspecies, Ae. aegypti aegypti and Ae. aegypti formosus, have been described based on body coloration. These subspecies remarkably differ from each other in their worldwide distribution, association with humans, and ability to transmit pathogens. Based on our preliminary data, we hypothesize that chromosomal inversions contribute to the establishment and maintenance of genomic and phenotypic divergence in aedini mosquitoes. Polymorphic inversions are often responsible for epidemiologically important phenotypes in Anopheles populations but they have never been directly observed in Aedes. Our long-term goal is to understand the underlying genomic determinants of epidemiologically important phenotypic and behavioral differences of the Ae. aegypti subspecies. Toward this end, we propose three specific aims for this project: 1) improve the genome assembly and gene annotation of Ae. aegypti based on Pacific Biosciences RS sequencing data; 2) develop a high- resolution physical map for the Ae. aegypti genome; 3) use the new genome assembly as a reference to detect inversions in 2 subspecies of Ae. aegypti and in a sister taxon, Ae. mascarensis. The innovative strategy of integrating a powerful 3rd generation sequencing technology with physical chromosome mapping will create an improved high-quality genome assembly that will make possible the discovery of chromosomal inversions in Ae. aegypti and will stimulate future genetic studies aimed at preventing mosquito- borne disease transmission.